Culture of retinal capillary endothelial cells

The culture of retinal capillary endothelial cells involves certain problems concerning contamination by pericytes, the maintenance of differentiation and the duration of culture viability. A procedure for the isolation and culture of capillary endothelial cells from bovine retina which overcomes these difficulties, is described. Microvessel fragments isolated by mechanical dispersion and filtration techniques adhere strongly to dishes coated with extracellular matrix produced by bovine corneal endothelial cells. The first migrating cells emerge from the original microvessel fragments two days after plating. This technique and subsequent cloning provides migrating and proliferating cells derived only from the retinal capillaries and uncontaminated by other cell types such as pericytes. Endothelial cells were grown on gelatin coated dishes in a serum supplemented medium (10% calf serum). Cell proliferation was significantly enhanced by the addition of basic fibroblast growth factor (1 ng/ml) to the culture medium. In these culture conditions, retinal capillary endothelial cells can be repeatedly passing without the loss of their principal morphological characteristics and some of the differentiated properties of endothelial cells. Primary cultures and subcultures, at least up to the 8th passage, formed a monolayer of small, elongated, tightly-packed, contact inhibited cells which expressed Factor VIII-related antigen. Ultrastructural examination by transmission electron microscopy of confluent bovine retinal capillary endothelial cells showed many tight junctions and Webel Palade granules. These studies provide new means for the isolation and culture of retinal capillary endothelial cells and presents evidence for growth factor requirements for the ability of cells to be repeatedly passing.     

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL. METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM). Glucose and aminoguanidine were then removed by dialysis. Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media. Cell counts were determined by hemocytometer. RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001). Aminoguanidine was as effective at 1 microM as at the higher concentrations. Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity. Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability. CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment. This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy.     

Interaction of retinal pericytes, capillary endothelial cells and pigment epithelial cells in vasoproliferative retinopathies

Degeneration of intramural pericytes is the first histopathological lesion seen in diabetic retinopathy. Since pericytes from the optic nerve and brain are spared, such degeneration appears to be specific to the retinal circulation. Recent studies have shown that when retinal pericytes and microvascular endothelial cells are co-cultured in vitro , direct contact between the two types of cells leads to the inhibition of proliferation of endothelial cells. Previously it was shown that when pericytes are cultured in the absence of microvascular endothelial cells, the cultured medium conditioned by the pericytes is actively mitogenic for retinal capillary endothelial cells. Furthermore, conditioned medium from human retinal pigment epithelial cells is also mitogenic for retinal capillary endothelial cells and pericytes. On the basis of these results, a new working hypothesis is proposed for the mechanism of action of photocoagulation used in vasoproliferative retinopathies.     

Interaction of retinal pericytes, capillary endothelial cells and pigment epithelial cells in vasoproliferative retinopathies

Degeneration of intramural pericytes is the first histopathological lesion seen in diabetic retinopathy. Since pericytes from the optic nerve and brain are spared, such degeneration appears to be specific to the retinal circulation. Recent studies have shown that when retinal pericytes and microvascular endothelial cells are co-cultured in vitro, direct contact between the two types of cells leads to the inhibition of proliferation of endothelial cells. Previously it was shown that when pericytes are cultured in the absence of microvascular endothelial cells, the cultured medium conditioned by the pericytes is actively mitogenic for retinal capillary endothelial cells. Furthermore, conditioned medium from human retinal pigment epithelial cells is also mitogenic for retinal capillary endothelial cells and pericytes. On the basis of these results, a new working hypothesis is proposed for the mechanism of action of photocoagulation used in vasoproliferative retinopathies.     

Impaired function of circulating CD34 CD45 cells in patients with proliferative diabetic retinopathy

Proliferative diabetic retinopathy is a consequence of retinal ischemia due to capillary occlusion resulting from damage to the retinal microvascular endothelium. Recent evidence suggests that high levels of bone-marrow derived circulating endothelial progenitor cells (EPCs) contribute to the pathological neovascularization of ischemic tissues and are a critical risk factor for the development of these complications. In the absence of a consensus definition of a circulating EPC and its surface markers in humans we evaluated the functional properties of CD34⁺ CD45⁻ endothelial colony forming cells (ECFCs) in patients with proliferative diabetic retinopathy (PDR). Higher levels of circulating CD34⁺ CD45⁻ cells were observed in patients with PDR compared to controls. However, ECFCs from patients with PDR were impaired in their ability to migrate towards SDF-1 and human serum, incorporate into and form vascular tubes with human retinal endothelial cells. The results from these pilot studies suggest that ECFCs from patients with PDR are mobilized into the circulation but may be unable to migrate and repair damaged capillary endothelium. This suggests that ECFCs may be a potential therapeutic target in the prevention and treatment of diabetic vascular complications.     

Simplified methods for consistent and selective culture of bovine retinal endothelial cells and pericytes

Different matrix components, in combination with various media and serum supplements, were evaluated for their ability to promote selectively the growth of bovine retinal endothelial cells in primary culture. The optimal setting for the selective growth of retinal endothelial cells was a fibronectin/hyaluronic acid matrix, Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% pooled human serum and 100 micrograms/ml heparin. These conditions consistently yielded virtually homogeneous cultures of endothelial cells, assessed using specific endothelial markers. Thus obtained, the retinal endothelial cells could be subcultured and maintained in phenotypically stable long-term serial cultivation. Homogeneous cultures of retinal pericytes were obtained when microvessel isolates were seeded to uncoated or gelatin-coated culture dishes and grown in DMEM supplemented with 20% fetal bovine serum. The retinal pericytes could also be subcultured and cultivated for numerous populations doublings. Additionally, observations from this study suggest that two populations of pericytes may be obtained in culture and distinguished on the basis of their relative size and antigenic properties.     

Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism

BACKGROUND: The success of long-term corneal organ culture is limited by the progressive loss of endothelial cells during culture and the use of culture medium supplemented with fetal calf serum as a possible source of contamination with infectious agents. In this study, we investigated the suitability of a serum-free medium (Endothelial-SFM) to improve preservation conditions for human donor corneas. METHODS: Six pairs of corneas were stored in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum (FCS) for 8-14 days. One cornea of each pair was then further cultivated in Endothelial-SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In a second series of experiments, the endothelial cell density of seven pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS or serum-free Endothelial-SFM. RESULTS: After precultivation in conventional medium, the endothelial cell density of corneas allocated to cultivation in Endothelial-SFM was 1000-1950 cells/mm2 and that of those subsequently cultured in MEM 1200-2000 cells/mm2. At 9 weeks, cell densities of 900-1500 cells/mm2 were found after cultivation in Endothelial-SFM compared with a total cell loss in MEM. Freshly isolated corneas cultured in Endothelial-SFM with or without FCS supplementation showed a decrease of endothelial cell density of about 20% within the first 2 weeks of storage. During further cultivation cell density remained constant without statistically significant differences between the groups. Glucose consumption of the corneas was higher in Endothelial-SFM than in MEM. Corneas stored in Endothelial-SFM with 2% FCS showed a higher glucose consumption than those preserved in serum-free Endothelial-SFM. CONCLUSION: Organ culture of human donor corneas using the serum-free basal medium Endothelial-SFM is superior to conventional culture conditions because the decrease in endothelial cell density can be ameliorated, the culture period can be prolonged and the risk of transmitting infectious agents via serum can be minimised.     

Role of mitochondrial superoxide dismutase in the development of diabetic retinopathy

PURPOSE: Apoptosis of retinal capillary cells is an early event in the pathogenesis of retinopathy in diabetes, and oxidative stress has been linked to accelerated apoptosis of retinal capillary cells. Mitochondria are the major endogenous source of superoxide, and superoxide is considered to be a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes. The purpose of the present study is to determine the role of mitochondrial superoxide dismutase (MnSOD) in the development of diabetic retinopathy. METHODS: The effect of overexpression of MnSOD on glucose-induced endothelial cell oxidative stress, nitrosative stress, and apoptosis was determined by using bovine retinal endothelial cells. Furthermore, the effect of diabetes in rats (11 months' duration) on the activity and the mRNA expression of retinal MnSOD were also determined. RESULTS: MnSOD activity in the nontransfected control retinal endothelial cells was 20% compared with the total SOD activity and was increased to 60% in the MnSOD-transfected cells. MnSOD overexpression prevented a glucose-induced increase in oxidative stress (8-hydroxy guanosine levels), nitrosative stress (nitrotyrosine formation), and apoptosis of retinal endothelial cells. MnSOD enzyme activity and its mRNA were decreased significantly in the retina obtained from the diabetic rats, and these abnormalities were prevented by long-term lipoic acid therapy. CONCLUSIONS: The results of this study suggest a protective role for MnSOD in retinal capillary cell death and, ultimately, in the pathogenesis of retinopathy in diabetes. Understanding the role of MnSOD to modify the course of retinopathy could elucidate important molecular targets for future pharmacological interventions.     

Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-Induced Retinopathy

Purpose. EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. Methods. Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. Results. EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. Conclusions. EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.     

鼠视网膜血管内皮细胞体外培养的改良及鉴定

背景视网膜血管内皮细胞（RVECs）的体外培养是进行视网膜血管性疾病研究的基础,人眼球供体的缺乏是限制人血管内皮细胞培养的主要原因,与人高度同源的大鼠血管内皮细胞的培养方法已经建立,但其分离方法对细胞损伤较大,影响细胞的培养效率。目的建立和改良大鼠RVECs的培养方法。方法选取5只SD大鼠眼球,获得视网膜,用组织块培养法,将大鼠视网膜用质量分数0．1％胶原酶消化,并加入质量分数20％胎牛血清、血管内皮生长因子（VEGF）、血管内皮细胞生长添加物（ECGS）,用血管内皮细胞培养基中和后充分吹打,制备细胞及组织悬液,接种于人纤维粘连蛋白（FN）包被的培养瓶中。用Ⅷ因子相关抗体鉴定培养的内皮细胞。结果组织块法消化培养2d可见细胞从组织块游出,培养4d可伸出触角呈梭形,5～6d可融合生长,培养14d后的细胞呈单层生长,培养的细胞Ⅷ因子相关抗体染色阳性。传代培养的细胞接种2h可重新贴壁,恢复融合生长状态。结论视网膜组织块培养并用胶原酶消化可获得活性较强的细胞和碎片,再用高选择性的血管内皮细胞培养基和FN促贴壁进行选择性培养,可获得纯度较高的RVECs。     

hsa_circ_0005699在高糖培养的人视网膜血管内皮细胞中的表达

目的 检测在高糖培养条件下人视网膜血管内皮细胞中hsa_circ_0005699的表达,为糖尿病性视网膜病变发病机制研究提供新的线索.方法 人视网膜血管内皮细胞分为低糖组(L组)、高渗对照组(C组)和高糖组(H组),设置低糖组中糖浓度为5.5mmol/L,高渗对照组中葡萄糖浓度为5.5mmol/L,甘露醇浓度为19.5...     

VEGF increases retinal vascular ICAM-1 expression in vivo.

PURPOSE: Intraocular injections of vascular endothelial growth factor (VEGF), a peptide implicated in the pathogenesis of diabetic retinopathy, can induce retinal ischemia. Diabetic retinal ischemia may be caused, in part, by the adhesion of leukocytes to the retinal vasculature. In this study, the ability of VEGF to increase the expression of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in capillary endothelium and the retinal vasculature was examined. METHODS: The expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin on human brain capillary endothelial cell monolayers exposed to VEGF was quantitated by immunoassay. The effect of VEGF on retinal vascular ICAM-1 expression was determined in ICAM-1 immunofluorescence studies of retinal flat-mounts and in RNase protection assays. RESULTS: VEGF increased capillary endothelial cell ICAM-1 levels in a dose- and time-dependent manner (6-24 hours, plateau after 6 hours; EC50, 25 ng/ml). VEGF failed to alter E-selectin, P-selectin, or VCAM-1 levels under the conditions tested. Intravitreal injections of pathophysiologically relevant concentrations of VEGF increased ICAM-1 protein and mRNA levels in the retinal vasculature. CONCLUSIONS: VEGF increases retinal vascular ICAM-1 expression. VEGF-induced increases in ICAM-1 may promote retinal leukostasis in diabetic eyes.     

Diabetic retinopathy. Pathophysiology and therapy of hypoxia-induced inflammation

Diabetic retinopathy is the most common chronic microvascular complication associated with diabetes mellitus. The development of diabetic retinopathy is a consequence of metabolic dysregulation. Hyperglycemia is a critical factor which is involved in basement membrane thickening, loss of pericytes and endothelial cells, and retinal capillary nonperfusion. We review the molecular basis of diabetic retinopathy and maculopathy and elaborate the role of growth factors and cytokines in the development of diabetic vascular alterations, their specific influence on the cellular interaction between retinal endothelial cells and pericytes, and the role of intravascular blood components.     

Pancreatic islet transplantation ameliorates the effect of diabetes on retinal capillary endothelial cell kinetics

Perturbations in capillary endothelial cell function are early events in the pathogenesis of diabetic retinopathy. We used tritiated thymidine autoradiography to study the turn-over rate of retinal capillary endothelial cells in retinovascular preparations from normal, untreated diabetic and diabetic mice treated with pancreatic islet-cell transplants. Capillary endothelial cell turnover was significantly increased in poorly controlled diabetes and this increase was reduced by islet-cell transplantation. The prevention of excessive endothelial cell replication by tight control of hyperglycemia may help prevent the characteristic structural changes of diabetic microangiopathy, such as basement membrane accumulation.     

糖尿病对血-视网膜屏障超微结构影响的研究进展

糖尿病视网膜病变（diabeticretinopathy,DR）是全世界最主要的致盲性眼病,也是最严重和最常见的微血管病变之一。糖尿病可造成血一视网膜屏障的损害引起血管源性水肿和神经组织损伤,造成视力下降。内层血-视网膜屏障主要是由视网膜毛细血管内皮细胞的紧密连接构成,此屏障阻碍血液的渗透及内源性物质和外源性物质在视网膜中的自由扩散,使视网膜保持恒定的环境,有效的供应营养物质。糖尿病患者的视网膜中由于细胞因子、生长因子、晚期糖基化终产物、炎症、高血糖症和周细胞丢失的增加,导致视网膜血管内皮细胞通透性增加。本文就糖尿病所引起的血-视网膜屏障超微结构改变进行综述。     

Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay

BACKGROUND: To evaluate the ability of different commercially available cell culture media to induce proliferation and morphological changes in primary cultures of human corneal endothelial cells (HCEC). This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas. METHODS: A total of 11 different culture media enriched with 0%, 2%, 5%, and 10% fetal calf serum (FCS) were compared. The test media were divided into three groups: Group 1: Media based on minimal essential medium (MEM), currently used for long-term corneal organ culture in European eye banks; Group 2: F99-based media, enriched for growth of corneal endothelial cells at serum-reduced conditions; and Group 3: Media designed for growth of special cell types or for short-term corneal organ culture. The growth-promoting capacity of each test medium was quantified using an HCEC proliferation assay, whereas changes in cell morphology were evaluated by phase-contrast microscopy. RESULTS: The morphological characteristics of HCEC were best maintained in the group of F99-based media, which also induced the highest level of cell proliferation under serum-reduced conditions. Specifically, the medium F99-Sr (F99 enriched with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids) induced a two- to three-fold higher HCEC density at both 0% and 2% FCS when compared to all other test media, and it also maintained the most endothelial cell-like morphology. Also, at higher serum concentrations (5% and 10% FCS), the cell growth was most prominent in F99-Sr, as well as in the medium SFM that originally was designed for serum-free growth of vascular endothelial cells. CONCLUSION: This study suggests that the media F99-Sr and SFM should be further tested and refined as potential new storage solutions for long-term corneal organ culture at physiological temperatures.     

Suppressed expression of tubedown-1 in retinal neovascularization of proliferative diabetic retinopathy.

PURPOSE: Retinal neovascularization occurring as a complication of diabetes mellitus can cause vision loss and blindness. The identification and study of novel genes involved in retinal angiogenesis may define new targets to suppress retinal neovascularization in diabetes and other ocular diseases. A novel acetyltransferase subunit, tubedown-1 (tbdn-1), has been isolated, the expression of which is regulated during blood vessel development. Tbdn-1 is not detected in most adult vascular beds but persists at high levels in the adult ocular vasculature. The purpose of this study was to gain insight into the possible role of tbdn-1 in retinal blood vessels by characterizing its expression patterns in adult homeostasis and in retinal neovascularization associated with diabetes. METHODS: Western blot analysis and immunohistochemistry were performed to study the expression patterns of tbdn-1 during adult homeostasis in normal human retinas, in a model of choroid-retina endothelial capillary outgrowth in vitro, and in retinas showing neovascularization in patients with proliferative diabetic retinopathy (PDR). RESULTS: In adults during homeostasis, tbdn-1 was expressed highly in normal endothelium of retinal and limbic blood vessels. Tbdn-1 was also expressed in RF/6A, a rhesus macaque choroid-retina-derived endothelial cell line. In an in vitro model system using the RF/6A cell line, tbdn-1 expression was downregulated during the outgrowth of these cells into capillary-like structures on a reconstituted basement membrane matrix. Similar to this in vitro model, tbdn-1 expression is specifically suppressed in the endothelial cells of blood vessels and capillary fronds in vivo in both the neural retinal tissue and in preretinal membranes in eyes of patients with PDR. CONCLUSIONS: High levels of expression of tbdn-1 are associated with ocular endothelial homeostasis in adults. Conversely, low levels of tbdn-1 expression are associated with endothelial capillary outgrowth in vitro and retinal neovascularization in vivo. Because the tbdn-1 acetyltransferase subunit is a member of a family of regulatory enzymes that are known to control a range of processes, including cell growth and differentiation, through posttranslational modification, the current results support a hypothesis that tbdn-1 may be involved in maintaining homeostasis and preventing retinal neovascularization.     

Aldose reductase activity in retinal and cerebral microvessels and cultured vascular cells

Isolated microvessels (primarily capillaries) from bovine retina and cerebral cortex, as well as cultured bovine retinal capillary pericytes and porcine and canine retinal capillary endothelial cells contain apparent aldose reductase activity. This conclusion is based on the ability of these cultured cells and vessel fragments to reduce DL-glyceraldehyde in preference to D-glucuronate at low (0.1 mM) substrate concentrations, in the presence of NADPH, and in the accumulation of high levels of sorbitol or galactitol when retinal pericytes and endothelial cells are cultured in media enriched in glucose or galactose. The quantitative similarities of these activities in bovine retinal and cerebral microvessels, as well as the quantitatively similar ability of these two sets of microvessels to oxidize 14C-labeled glucose with the label either in the C-1 or the C-6 position, suggests that aldose reductase may not be a major causal factor in diabetic retinopathy. This conclusion is suggested because, while these metabolic activities are similar in bovine retinal and cerebral microvessels, only the retinal microvasculature suffers major anatomic and functional damage in diabetes. This conclusion must be viewed with caution, however, because other metabolic pathways that we have not investigated may be altered by an excess of sugar alcohols, and be present in differing activities in retinal and cerebral microvessels; species differences may exist; and similar experiments have not been conducted using human microvessels.